A perennial goal in the pharmacological arts has been the development of methods and compositions to facilitate the specific delivery of therapeutic and other agents to the appropriate cells and tissues that would benefit from such treatment, and the avoidance of the general physiological effects of the inappropriate delivery of such agents to other cells or tissues of the body. Recently, the advent of recombinant DNA technology and genetic engineering has provided the pharmacological arts with a wide new spectrum of agents that are functional genes carried in recombinant expression constructs capable of mediating expression of these genes in host cells. These developments have carried the promise of xe2x80x9cmolecular medicinexe2x80x9d, specifically gene therapy, whereby a defective gene could be replaced by an exogenous copy of its cognate, functional gene, thereby alleviating a variety of genetic diseases.
However, the greatest drawback to the achievement of effective gene therapy has been the inability in the art to introduce recombinant expression constructs encoding functional eukaryotic genes into cells and tissues in vivo. While it has been recognized in the art as being desirable to increase the efficiency and specificity of administration of gene therapy agents to the cells of the relevant tissues, the goal of specific delivery has not bee achieved in the prior art.
Liposomes have been used to attempt cell targeting. Rahman et al., 1982, Life Sci. 31: 2061-71 found that liposomes which contained galactolipid as part of the lipid appeared to have a higher affinity for parenchymal cells than liposomes which lacked galactolipid. To date, however, efficient or specific delivery has not been predictably achieved using drug-encapsulated liposomes. There remains a need for the development of a cell- or tissue-targeting delivery system.
Thus there remains in the art a need for methods and reagents for achieving cell and tissue-specific targeting of gene therapy agents, particularly recombinant expression constructs encoding functional genes, in vivo.
The present invention is directed to an improved methods for targeted delivery of functional eukaryotic genes to cells and tissues in vivo. This delivery system achieves such specific delivery by the formation of DNA:lipid complexes between nucleic acid comprising a recombinant expression construct encoding a functional eukaryotic gene or fragment thereof complexed with a mixture of a cationic lipid and a neutral lipid. Methods of use are also provided. This invention has the specific advantage of targeted delivery of functional eukaryotic genes into cells in vivo, achieving effective intracellular delivery of constructs encoding functional genes more efficiently and with more specificity than conventional delivery systems.
In a fist embodiment, the invention provides a pharmaceutical composition, comprising a formulation of a soluble complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid in a pharmaceutically acceptable carrier suitable for administration to an animal by injection. In these embodiments of the invention, the recombinant expression construct comprises a nucleic acid encoding a protein, the nucleic acid being operatively linked to gene expression regulatory elements and whereby the protein encoded by the nucleic acid is expressed.
In this first embodiment, the cationic lipid is a nitrogen-containing, imidazolium-derived cationic lipid having the formula: 
wherein each of R and R1 independently is a straight-chain, aliphatic hydrocarbyl group of 11 to 29 carbon atoms inclusive. Preferred are those cations wherein each of R and R1independently have from 13 to 23 carbon atoms inclusive. In particularly preferred embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole. In additional preferred embodiments, the neutral lipid is cholesterol, and the 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole and cholesterol are present in the complex at a ratio of 1:1. Further preferred embodiments comprise a recombinant expression construct encoding human CFTR and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of from about 1:6 to about 1:8 (xcexcgDNA:nmoles lipid). Particularly preferred are embodiments where the DNA comprising the recombinant expression construct is present in the complex at a concentration of about 0.5 to 1 mg/mL. In further preferred embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole and the neutral lipid is dioleoylphosphatidylethanolamine, and the 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole and dioleoylphosphatidylethanolamine are present in the complex at a ratio of 1:1. Further preferred embodiments comprise a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of about 1:1. Particularly preferred are embodiments where the DNA comprising the recombinant expression construct is present in the complex at a concentration of about 0.5 to 5mg/mL.
In a second embodiment, the invention provides methods for introducing a recombinant expression construct into a cell comprising lung tissue in an animal, the method comprising the step of administering the pharmaceutical composition of claim 1 to the animal by intravenous injection. In preferred embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole. In additional preferred embodiments, the neutral lipid is cholesterol, and the 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole and cholesterol are present in the complex at a ratio of 1:1. Further preferred embodiments comprise a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of from about 1:6 to about 1:8. Particularly preferred are embodiments where the DNA comprising the recombinant expression construct is present in the complex at a concentration of about 0.5-1 mg/mL.
In another aspect of the second embodiment of the invention is provided methods for introducing a recombinant expression construct into a cell comprising spleen tissue in an animal, the method comprising the step of administering the pharmaceutical composition of claim 1 to the animal by intravenous injection. In preferred embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole. In additional preferred embodiments, the neutral lipid is dioleoylphosphatidylethanolamine, and the cationic lipid and the neutral lipid are present in a ratio of 1:1. Further preferred embodiments comprise a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of about 1:1. Particularly preferred are embodiments where the DNA comprising the recombinant expression construct is present in the complex at a concentration of about 1-2.5 mg/mL.
In further embodiments, the DNA:lipid complex is targeted to peritoneal macrophages by administration by intraperitoneal injection. In these embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole, the neutral lipid is cholesterol, the cationic lipid and the neutral lipid are present in a ratio of about 1:1, the complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of about 1:1, the DNA concentration in the DNA:lipid complexes is about 1-2.5 mg/mL. In additional embodiments of this aspect of the invention, the DNA:lipid complex is targeted to spleen macrophages and administered by intraperitoneal injection. In these embodiments, the cationic lipid is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl) imidazole, the neutral lipid is cholesterol, the cationic lipid and the neutral lipid are present in a ratio of about 1:1, the complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of about 1:1, the DNA concentration in the DNA:lipid complexes is about 1 to 2.5 mg/mL.
In this aspect, the invention also provides methods for targeting gene transfer into pancreatic tissue by intraperitoneal injection. In preferred embodiments, the cationic lipid is is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole, the neutral lipid is dioleoylphosphatidylethanolamine, the cationic lipid and the neutral lipid are present in a ratio of about 1:1, the complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprises a ratio of DNA to lipid of about 1:1, the DNA concentration in the DNA:lipid complexes is about 1.5 to about 2.5 mg/mL.
The invention also provides a method of introducing a recombinant expression construct into a cell comprising a tissue in an animal, the method comprising the step of administering the pharmaceutical composition of Claim 1 to the animal by direct injection. In preferred embodiments, the cationic lipid is is 1-(2-(oleoyloxy)ethyl)-2-oleyl-3-(2-hydroxyethyl)imidazole and the neutral lipid is cholesterol. Also preferred are mixtures of the cationic lipid and the neutral lipid in a ratio of about 1:1. Preferred complexes include a complex of a recombinant expression construct and a mixture of a neutral lipid and a cationic lipid comprising a ratio of DNA to lipid of about 1:1. The preferred DNA concentration in the DNA:lipid complexes is about 1-2.5 mg/mL in this embodiment of the invention.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.